Method of improving the properties of a flour dough, a flour dough improving composition and improved food products

ABSTRACT

A method of improving the rheological properties of a flour dough and the quality of the finished product made from such a dough, comprising adding an effective amount of an oxidoreductase capable of oxidizing maltose, in particular a hexose oxidase, e.g. isolated from an algal species such as  Iridophycus flaccidum, Chondrus crispus  or  Euthora cristata  and a dough improving composition comprising the oxidoreductase.

FIELD OF INVENTION

[0001] The invention pertains to the provision of flour doughs havingimproved rheological properties and farinaceous food products havingimproved quality characteristics and it provides a maltose oxidizingoxidoreductase-containing composition capable of conferring suchimproved properties on doughs and finished food products made herefromwhen it is added as a component to the doughs, and a method of preparingimproved doughs and farinaceous food products.

TECHNICAL BACKGROUND AND PRIOR ART

[0002] The invention relates in particular to a method of providingflour doughs having improved rheological properties and to finishedbaked or dried products made from such doughs, which have improvedtextural, eating quality and dimensional characteristics.

[0003] In this connection, the “strength” or “weakness” of doughs is animportant aspect of making farinaceous finished products from doughs,including baking. The “strength” or “weakness” of a dough is primarilydetermined by its content of protein and in particular the content andthe quality of the gluten protein is an important factor in thatrespect. Flours with a low protein content is generally characterized as“weak”. Thus, the cohesive, extensible, rubbery mass which is formed bymixing water and weak flour will usually be highly extensible whensubjected to stress, but it will not return to its original dimensionswhen the stress is removed.

[0004] Flours with a high protein content are generally characterized as“strong” flours and the mass formed by mixing such a flour and waterwill be less extensible than the mass formed from a weak flour, andstress which is applied during mixing will be restored without breakdownto a greater extent than is the case with a dough mass formed from aweak flour. Strong flour is generally preferred in most baking contextsbecause of the superior rheological and handling properties of the doughand the superior form and texture qualities of the finished baked ordried products made from the strong flour dough.

[0005] Doughs made from strong flours are generally more stable.Stability of a dough is one of the most important characteristics offlour doughs. According to American Association of Cereal Chemists(AACC) Method 36-01A the term “stability” can be defined as “the rangeof dough time over which a positive response is obtained and thatproperty of a rounded dough by which it resists flattening under its ownweight over a course of time”. According to the same method, the term“response” is defined as “the reaction of dough to a known and specificstimulus, substance or set of conditions, usually determined by bakingit in comparison with a control”.

[0006] Within the bakery and milling industries it is known to use dough“conditioners” to strengthen the dough. Such dough conditioners arenormally non-specific oxidizing agents such as eg iodates, peroxides,ascorbic acid, K-bromate or azodi-carbonamide and they are added todough with the aims of improving the baking performance of flour toachieve a dough with improved stretchability and thus having a desirablestrength and stability. The mechanism behind this effect of oxidizingagents is that the flour proteins, in particular gluten contains thiolgroups which, when they become oxidized, form disulphide bonds wherebythe protein forms a more stable matrix resulting in a better doughquality and improvements of the volume and crumb structure of the bakedproducts.

[0007] In addition to the above usefulness of ascorbic acid/ascorbate asa dough conditioner due to its oxidizing capacity, these compounds mayalso act as substrate for an oxidoreductase, ascorbate oxidase which isdisclosed in EP 0 682 116 A1. In the presence of its substrate, thisenzyme converts ascorbic acid/ascorbate to dehydroascorbic acid andH₂O₂. This prior art does not suggest that ascorbic acid oxidase in thepresence of ascorbic acid/ascorbate might have a dough conditioningeffect, but assumingly this is the case.

[0008] However, the use of several of the currently available oxidizingagents is either objected to by consumers or is not permitted byregulatory bodies and accordingly, it has been attempted to findalternatives to these conventional flour and dough additives and theprior art has i.a. suggested the use of glucose oxidase for thispurpose.

[0009] Thus, U.S. Pat. No. 2,783,150 discloses the addition of glucoseoxidase to flour to improve dough strength and texture and appearance ofbaked bread.

[0010] CA 2,012,723 discloses bread improving compositions comprisingcellulolytic enzymes such as xylanases and glucose oxidase, the latterenzyme being added to reduce certain disadvantageous effects of thecellulolytic enzymes (reduced dough strength and stickiness) and it isdisclosed that addition of glucose to the dough is required to obtain asufficient glucose oxidase activity.

[0011] JP-A-92-084848 suggests the use of a bread improving compositioncomprising glucose oxidase and lipase.

[0012] EP-B1-321 811 discloses the use of an enzyme compositioncomprising sulfhydryl oxidase and glucose oxidase to improve therheological characteristics of doughs. It is mentioned in this prior artdocument that the use of glucose oxidase alone has not been successful.

[0013] In EP-B1-338 452 is disclosed an enzyme composition for improvingdough stability, comprising a mixture of cellulase/hemicellulase,glucose oxidase and optionally sulfhydryl oxidase.

[0014] However, the use of glucose oxidase as a dough improving additivehas the limitation that this enzyme requires the presence of sufficientamounts of glucose as substrate in order to be effective in a doughsystem and generally, the glucose content in cereal flours is low.Therefore, the absence of glucose in doughs or the low content hereof indoughs will be a limiting factor for the effectiveness of glucoseoxidase as a dough improving agent.

[0015] In contrast hereto, the content of maltose in cereal flours isgenerally significantly higher than that of glucose and accordingly, afreshly prepared dough will normally contain more maltose than glucose.Thus, in an experiment where the content of sugars in supernatants fromsuspensions of wheat flour and a dough prepared from the flour andfurther comprising water, yeast, salt and sucrose (as described in thefollowing example 2.3) were analyzed, the following values (% by weightcalculated on flour) were found: Flour Dough Sucrose 0.3 <0.01 Galactose0.001 0.01 Glucose 0.25 0.72 Maltose 2.6 1.4 Fructose 0.008 0.67 Lactose<0.01 <0.01

[0016] In addition, the content of maltose remains at a relatively highlevel in a dough which is leavened by yeast, since the yeast primarilyutilizes glucose, or it may even increase in the dough e.g. duringproofing due to the activity of starch degrading enzymes such as e.g.β-amylase, which is inherently present in the flour or is added to thedough.

[0017] Whereas the prior art has recognized the useful improving effectsof glucose oxidase on the rheological characteristics of bread doughsand on the quality of the corresponding baked products, it has also beenrealized that the use of this enzyme has several drawbacks. Thus, it maybe required to add sucrose or glucose as substrate to the dough toobtain a sufficient effect and glucose oxidase does not constantlyprovide a desired dough or bread improving effect when used alonewithout the addition of other enzymes.

[0018] However, it has now been found that the addition of anoxidoreductase, which is capable of oxidizing maltose, including hexoseoxidase as a sole dough conditioning agent, i.e. without concomitantaddition of substrate for the added enzyme, or of other enzymes, to afarinaceous dough results in an increased resistance hereof to breakingwhen the dough is stretched, i.e. this enzyme confers in itself to thedough an increased strength whereby the dough becomes less prone tomechanical deformation. It is contemplated that this effect of additionof hexose oxidase to a dough is the result of the formation ofcross-links between thiol groups in sulphur-containing amino acids inwheat gluten which occurs when the H₂O₂ generated by the enzyme in thedough reacts with the thiol groups which are hereby oxidized.

[0019] Hexose oxidase (D-hexose:O₂-oxidoreductase, EC 1.1.3.5) is anenzyme which in the presence of oxygen is capable of oxidizing D-glucoseand several other reducing sugars including maltose, glucose, lactose,galactose, xylose, arabinose and cellobiose to their correspondinglactones with subsequent hydrolysis to the respective aldobionic acids.Accordingly, hexose oxidases differ from glucose oxidase which can onlyconvert D-glucose, in that hexose oxidases can utilize a broader rangeof sugar substrates. The oxidation catalyzed by the enzyme can beillustrated as follows:

D-Glucose+O₂→δ-D-gluconolactone+H₂O₂,

[0020] or

D-Galactose+O₂→γ-D-galactogalactone+H₂O₂

[0021] Hexose oxidase (in the following also referred to as HOX) hasbeen isolated from several red algal species such as Iridophycusflaccidum (Bean and Hassid, 1956, J. Biol. Chem., 218:425-436) andChondrus crispus (Ikawa 1982, Methods Enzymol., 89:145-149).Additionally, the algal species Euthora cristata (Sullivan et al. 1973,Biochemica et Biophysica Acta, 309:11-22) has been shown to produce HOX.

[0022] Other potential sources of hexose oxidase according to theinvention include microbial species or land-growing plant species. Thus,as an example of such a plant source, Bean et al., Journal of BiologicalChemistry (1961) 236: 1235-1240, have disclosed an oxidoreductase fromcitrus fruits which is capable of oxidizing a broad range of sugarsincluding D-glucose, D-galactose, cellobiose, lactose, maltose,D-2-deoxyglucose, D-mannose, D-glucosamine and D-xylose. Another exampleof an enzyme having hexose oxidase activity is the enzyme system ofMalleomyces mallei disclosed by Dowling et al., Journal of Bacteriology(1956) 72:555-560.

[0023] It has been reported that hexose oxidase isolated from the abovenatural sources may be of potential use in the manufacturing of certainfood products. Thus, hexose oxidase isolated from Iridophycus flaccidumhas been shown to be capable of converting lactose in milk with theproduction of the corresponding aldobionic acid and has been shown to beof potential interest as an acidifying agent in milk, e.g. to replaceacidifying microbial cultures for that purpose (Rand, 1972, Journal ofFood Science, 37:698-701). In that respect, hexose oxidase has beenmentioned as a more interesting enzyme than glucose oxidase, since thislatter enzyme can only be enzymatically effective in milk or other foodproducts not containing glucose or having a low content of glucose, ifglucose or the lactose-degrading enzyme lactase which convert thelactose to glucose and galactose, is also added.

[0024] The capability of oxidoreductases including that of hexoseoxidase to generate hydrogen peroxide has also been utilized to improvethe storage stability of certain food products including cheese, butterand fruit juice as it is disclosed in JP-B-73/016612. It has also beensuggested that oxidoreductases may be potentially useful as antioxidantsin food products.

[0025] However, the present invention has demonstrated that hexoseoxidase is highly useful as a dough conditioning agent in themanufacturing of flour dough products including not only bread productsbut also other products made from flour doughs such as noodles andalimentary paste products.

SUMMARY OF THE INVENTION

[0026] Accordingly, the invention relates in a first aspect to a methodof improving the rheological properties of a flour dough and the qualityof the finished product made from the dough, comprising adding to thedough ingredients, dough additives or the dough an effective amount ofan oxidoreductase which at least is capable of oxidizing maltose, suchas e.g. a hexose oxidase.

[0027] In a further aspect, there is also provided a dough bakeryproduct improving composition comprising an oxidoreductase which atleast is capable of oxidizing maltose, and at least one further doughingredient or dough additive.

[0028] In still further aspects, the invention pertains to a method ofpreparing a bakery product, comprising preparing a flour dough includingadding an effective amount of an oxidoreductase which at least iscapable of oxidizing maltose and baking the dough, and a method ofpreparing a dough-based food product comprising adding to the dough aneffective amount of a maltose oxidizing oxidoreductase.

DETAILED DISCLOSURE OF THE INVENTION

[0029] In one aspect, the present method contemplates a method ofimproving the rheological properties of flour doughs. The methodcomprises, as it is mentioned above, the addition of an effective amountof a maltose oxidizing oxidoreductase either to a component of the doughrecipe or to the dough resulting from mixing all of the components forthe dough. In the present context, “an effective amount” is used toindicate that the amount is sufficient to confer to the dough and/or thefinished product improved characteristics as defined herein.

[0030] In one useful embodiment of the method according to theinvention, the oxidoreductase is a hexose oxidase. Hexose oxidase can,as it is described in details herein, be isolated from marine algalspecies naturally producing that enzyme. Such species are found in thefamily Gigartinaceae which belong to the order Gigartinales. Examples ofhexose oxidase producing algal species belonging to Gigartinaceae areChondrus crispus and Iridophycus flaccidum. Also algal species of theorder Cryptomeniales including the species Euthora cristata arepotential sources of hexose oxidase.

[0031] When using such natural sources for hexose oxidase, the enzyme istypically isolated from the algal starting material by extraction usingan aqueous extraction medium. As starting material may be used algae intheir fresh state as harvested from the marine area where they grow, orthe algal material can be used for extraction of hexose oxidase afterdrying the fronds e.g. by air-drying at ambient temperatures or by anyappropriate industrial drying method such as drying in circulated heatedair or by freeze-drying. In order to facilitate the subsequentextraction step, the fresh or dried starting material may advantageouslybe comminuted e.g. by grinding or blending.

[0032] As the aqueous extraction medium, buffer solutions e.g. having apH in the range of 5-8, such as 0.1 M sodium phosphate buffer, 20 mMtriethanolamine buffer or 20 mM Tris-HCl buffer are suitable. The hexoseoxidase is typically extracted from the algal material by suspending thestarting material in the buffer and keeping the suspension at atemperature in the range of 0-20° C. such as at about 5° C. for 1 to 5days, preferably under agitation.

[0033] The suspended algal material is then separated from the aqueousmedium by an appropriate separation method such as filtration, sievingor centrifugation and the hexose oxidase is subsequently recovered fromthe filtrate or supernatant. Optionally, the separated algal material issubjected to one or more further extraction steps.

[0034] Since several marine algae contain coloured pigments such asphycocyanins, it may be required to subject the filtrate or supernatantto a further purification step whereby these pigments are removed. As anexample, the pigments may be removed by treating the filtrate orsupernatant with an organic solvent in which the pigments are solubleand subsequently separating the solvent containing the dissolvedpigments from the aqueous medium. Alternatively, pigments may be removedby subjecting the filtrate or supernatant to a hydrophobic interactionchromatography step.

[0035] The recovery of hexose oxidase from the aqueous extraction mediumis carried out by any suitable conventional methods allowing isolationof proteins from aqueous media. Such methods, examples of which will bedescribed in details in the following, include conventional methods forisolation of proteins such as ion exchange chromatography, optionallyfollowed by a concentration step such as ultrafiltration. It is alsopossible to recover the enzyme by adding substances such as e.g.(NH₄)₂SO₄ or polyethylene glycol (PEG) which causes the protein toprecipitate, followed by separating the precipitate and optionallysubjecting it to conditions allowing the protein to dissolve.

[0036] For certain applications of hexose oxidase it is desirable toprovide the enzyme in a substantially pure form e.g. as a preparationessentially without other proteins or non-protein contaminants andaccordingly, the relatively crude enzyme preparation resulting from theabove extraction and isolation steps may be subjected to furtherpurification steps such as further chromatography steps, gel filtrationor chromato-focusing as it will also be described by way of example inthe following.

[0037] In a preferred embodiment of the method according to theinvention, a flour dough is prepared by mixing flour with water, aleavening agent such as yeast or a conventional chemical leaveningagent, and an effective amount of hexose oxidase under dough formingconditions. It is, however, within the scope of the invention thatfurther components can be added to the dough mixture.

[0038] Typically, such further dough components include conventionallyused dough components such as salt, a sweetening agent such as sugars,syrups or artificial sweetening agents, lipid substances includingshortening, margarine, butter or an animal or vegetable oil and one ormore dough additives such as emulsifying agents, starch degradingenzymes, cellulose or hemicellulose degrading enzymes, proteases,lipases, non-specific oxidizing agents such as those mentioned above,flavouring agents, lactic acid bacterial cultures, vitamins, minerals,hydrocolloids such as alginates, carrageenans, pectins, vegetable gumsincluding e.g. guar gum and locust bean gum, and dietary fibersubstances.

[0039] Conventional emulsifiers used in making flour dough productsinclude as examples monoglycerides, diacetyl tartaric acid esters ofmono- and diglycerides of fatty acids, and lecithins e.g. obtained fromsoya. Among starch degrading enzymes, amylases are particularly usefulas dough improving additives. α-amylase breaks down starch into dextrinswhich are further broken down by β-amylase into maltose. Other usefulstarch degrading enzymes which may be added to a dough compositioninclude glucoamylases and pullulanases. In the present context, furtherinteresting enzymes are xylanases and other oxidoreductases such asglucose oxidase, pyranose oxidase and sulfhydryl oxidase.

[0040] A preferred flour is wheat flour, but doughs comprising flourderived from other cereal species such as from rice, maize, barley, ryeand durra are also contemplated.

[0041] The dough is prepared by admixing flour, water, theoxidoreductase according to the invention and other possible ingredientsand additives. The oxidoreductase can be added together with any doughingredient including the water or dough ingredient mixture or with anyadditive or additive mixture. The dough can be prepared by anyconventional dough preparation method common in the baking industry orin any other industry making flour dough based products.

[0042] The oxidoreductase can be added as a liquid preparation or in theform of a dry powder composition either comprising the enzyme as thesole active component or in admixture with one or more other doughingredients or additive. The amount of the enzyme component addednormally is an amount which results in the presence in the finisheddough of 1 to 10,000 units per kg of flour, preferably 5 to 5000 unitssuch as 10 to 1000 units. In useful embodiments, the amount is in therange of 20 to 500 units per kg of flour. In the present context 1oxidoreductase unit corresponds to the amount of enzyme which underspecified conditions results in the conversion of 1 μmole glucose perminute. The activity is stated as units per g of enzyme preparation.

[0043] The effect of the oxidoreductase on the rheological properties ofthe dough can be measured by standard methods according to theInternational Association of Cereal Chemistry (ICC) and the AmericanAssociation of Cereal Chemistry (AACC) including the amylograph method(ICC 126), the farinograph method (AACC 54-21) and the extensigraphmethod (AACC 54-10). The extensigraph method measures e.g. the doughsability to retain gas evolved by yeast and the ability to withstandproofing. In effect, the extensigraph method measures the relativestrength of a dough. A strong dough exhibits a higher and, in somecases, a longer extensigraph curve than does a weak dough. AACC method54-10 defines the extensigraph in the following manner: “theextensigraph records a load-extension curve for a test piece of doughuntil it breaks. Characteristics of load-extension curves orextensigrams are used to assess general quality of flour and itsresponses to improving agents”.

[0044] In a preferred embodiment of the method according to theinvention, the resistance to extension of the dough in terms of theratio between the resistance to extension (height of curve, B) and theextensibility (length of curve, C), i.e. the B/C ratio as measured bythe AACC method 54-10 is increased by at least 10% relative to that ofan otherwise similar dough not containing oxidoreductase. In morepreferred embodiments, the resistance to extension is increased by atleast 20%, such as at least 50% and in particular by at least 100%.

[0045] The method according to the invention can be used for any type offlour dough with the aims of improving the rheological properties hereofand the quality of the finished products made from the particular typeof dough. Thus, the method is highly suitable for the making ofconventional types of yeast leavened bread products including wheatflour based bread products such as loaves and rolls. However, it iscontemplated that the method also can improve the properties of doughsin which leavening is caused by the addition of chemical leaveningagents, including sweet bakery products such as cake products includingas examples pound cakes and muffins, or scones.

[0046] In one interesting aspect, the invention is used to improve therheological properties of doughs intended for noodle products including“white noodles” and “chinese noodles” and to improve the texturalqualities of the finished noodle products. A typical basic recipe forthe manufacturing of noodles comprises the following ingredients: wheatflour 100 parts, salt 0.5 parts and water 33 parts. The noodles aretypically prepared by mixing the ingredients in an appropriate mixingapparatus followed by rolling out the noodle dough using an appropriatenoodle-machine to form the noodle strings which are subsequently airdried.

[0047] The quality of the finished noodles is assessed ia by theircolour, cooking quality and texture. The noodles should cook as quicklyas possible, remain firm after cooking and should preferably not looseany solids to the cooking water. On serving the noodles shouldpreferably have a smooth and firm surface not showing stickiness andprovide a firm “bite” and a good mouthfeel. Furthermore, it is importantthat the noodles have a light colour.

[0048] Since the appropriateness of wheat flour for providing noodleshaving the desired textural and eating qualities may vary according tothe year and the growth area, it is usual to add noodle improvers to thedough in order to compensate for sub-optimal quality of the flour.Typically, such improvers will comprise dietary fiber substances,vegetable proteins, emulsifiers and hydrocolloids such as e.g.alginates, carrageenans, pectins, vegetable gums including guar gum andlocust bean gum, and amylases.

[0049] It has been attempted to use glucose oxidase as a noodleimproving agent. However, as mentioned above, the content of glucose maybe so low in wheat flour that this enzyme will not be effective.

[0050] It is therefore an important aspect of the invention that theoxidoreductase according to the invention is useful as a noodleimproving agent, optionally in combination with other componentscurrently used to improve the quality of noodles. Thus, it iscontemplated that noodles prepared in accordance with the above methodwill have improved properties with respect to colour, cooking and eatingqualities including a firm, elastic and non-sticky texture andconsistency.

[0051] In a further useful embodiment the dough which is prepared by themethod according to the invention is a dough for preparing an alimentarypaste product. Such products which include as examples spaghetti andmaccaroni are typically prepared from a dough comprising as the mainingredients flour and eggs. After mixing of the ingredient, the dough isformed to the desired type of paste product and air dried. It iscontemplated that the addition to a paste dough will have a significantimproving effect on the extensibility and stability hereof resulting infinished paste product having improved textural and eating qualities.

[0052] In a further aspect of the invention there is provided a doughimproving composition comprising the oxidoreductase according to theinvention and at least one further dough ingredient or dough additive.

[0053] In a preferred embodiment, the oxidoreductase is hexose oxidase.The further ingredient or additive can be any of the ingredients oradditives which are described above. The composition may conveniently bea liquid preparation comprising the oxidoreductase. However, thecomposition is conveniently in the form of dry composition. It will beunderstood that the amount of oxidoreductase activity in the compositionwill depend on the types and amounts of the further ingredients oradditives. However, the amount of oxidoreductase activity is preferablyin the range of 10 to 100,000 units, preferably in the range of 100 to50,000 units such as 1,000 to 10,000 units including 2,000 to 5,000units.

[0054] Optionally, the composition may be in the form of a completedough additive mixture or pre-mixture for a making a particular finishedproduct and containing all of the dry ingredients and additives for sucha dough. In specific embodiments, the composition may be oneparticularly useful for preparing a baking product or in the making of anoodle product or an alimentary paste product.

[0055] As mentioned above, the present invention provides a method forpreparing a bakery product including the addition to the dough of anoxidoreductase such as e.g. hexose oxidase. In particular, this methodresults in bakery products such as the above mentioned products in whichthe specific volume is increased relative to an otherwise similar bakeryproduct, prepared from a dough not containing oxidoreductase. In thiscontext, the expression “specific volume” is used to indicate the ratiobetween volume and weight of the product. It has surprisingly been foundthat in accordance with the above method, the specific volume can beincreased significantly such as by at least 10%, preferably by at least20%, including by at least 30%, preferably by at least 40% and morepreferably by at least 50%.

[0056] In one advantageous embodiment of the above method at least onefurther enzyme is added to the dough. Suitable examples hereof include acellulase, a hemicellulase, a xylanase, a starch degrading enzyme, aglucose oxidase, a lipase and a protease.

[0057] The invention will now be described by way of illustration in thefollowing non-limiting examples and the drawing in which

[0058] FIG. 1 illustrates the changes of the B/C ratio relative tocontrol dough without enzyme in a dough to which was added 100 units/kgflour of hexose oxidase (black columns) or 100 units/kg flour of glucoseoxidase (grey columns) after 45, 90 and 135 minutes, respectively asmeasured by the AACC extensigraph method 54-10.

EXAMPLE 1

[0059] 1.1. Purification of Hexose Oxidase from Chondrus crispus

[0060] A purified hexose oxidase preparation was obtained using thebelow extraction and purification procedures. During these proceduresand the following characterizations of the purified enzyme, thefollowing assay for determination of hexose oxidase activity was used:

[0061] 1.1.1. Assay of Hexose Oxidase Activity

[0062] The assay was based on the method described by Sullivan and Ikawa(Biochimica et Biophysica Acta, 1973, 309:11-22), but modified to run inmicrotiter plates. An assay mixture contained 150 μl β-D-glucose (0.1 Min 0.1 M sodium phosphate buffer, pH 6.3), 120 μl 0.1 M sodium phosphatebuffer, pH 6.3, 10 μl o-dianisidine-dihydrochloride (Sigma D-3252, 3.0mg/m in H₂O), 10 μl peroxidase (POD) (Sigma P-8125, 0.1 ml in 0.1 Msodium phosphate buffer, pH 6.3) and 10 μl enzyme (HOX) solution. Blankswere made by adding buffer in place of enzyme solution.

[0063] The incubation was started by the addition of glucose. After 15minutes of incubation at 25° C. the absorbance at 405 nm was read in anELISA reader. A standard curve was constructed using varyingconcentrations of H₂O₂ in place of the enzyme solution.

[0064] The reaction can be described in the following manner:

[0065] Oxidized o-dianisidine has a yellow colour absorbing at 405 nm.

[0066] 1.1.2. Extraction

[0067] Fresh Chondrus crispus fronds were harvested along the coast ofBrittany, France. This fresh material was homogenized in a pin mill(Alpine). To a 100 g sample of the resulting homogenized frond materialwas added 300 ml of 0.1 M sodium phosphate buffer, pH 6.8. The mixturewas subsequently sonicated in a sonication bath for 5 minutes and thenextracted under constant rotation for 4 days at 5° C., followed bycentrifugation of the mixture at 47,000×g for 20 minutes.

[0068] 300 ml of the resulting clear pink supernatant was desalted byultrafiltration using an Amicon ultrafiltration unit equipped with anOmega (10 kD cut off, Filtron) ultrafiltration membrane.

[0069] 1.1.3. Anion Exchange Step

[0070] The retentate resulting from 1.1.2 was applied to a 5×10 cmcolumn with 200 ml Q-Sepharose FF equilibrated in 20 mM triethanolamine,pH 7.3. The column was washed with the equilibration buffer and hexoseoxidase eluted with a 450 ml gradient of 0 to 1 M of NaCl inequilibration buffer. The column was eluted at 6 ml/minute, andfractions of 14 ml collected. Fractions 9-17 (total 125 ml) were pooledand concentrated by ultrafiltration using an Amicon 8400 unit equippedwith an Omega (10 kD cut off, Filtron) ultrafiltration membrane to 7.5ml.

[0071] 1.1.4. Gel Filtration

[0072] The above 7.5 ml retentate was applied to a Superdex 200 2.6×60cm gel filtration column equilibrated in 50 mM sodium phosphate buffer,pH 6.4 and eluted at a flow rate of 1 ml/minute. Fractions of 4 ml werecollected. Fractions 17-28 (total volume 50 ml) containing the hexoseoxidase activity were pooled.

[0073] 1.1.5. Hydrophobic Interaction Chromatography

[0074] To the pool resulting from the gel filtration step 1.1.4 ammoniumsulphate was added to a final concentration of 2 M. This mixture wasthen applied to a 1.6×16 cm column with 32 ml phenyl sepharoseequilibrated in 20 mM sodium phosphate buffer, pH 6.3 and 2 M (NH₄)₂SO₄.The column was washed with equilibration buffer followed by elution ofhexose oxidase at a flow rate of 2 ml/minute using a 140 linear gradientfrom 2 M to 0 M (NH₄)₂SO₄ in 20 mM sodium phosphate buffer. Fractions of4 ml were collected and fractions 24-33 containing the hexose oxidaseactivity were pooled.

[0075] The above mentioned pink colour accompanies the enzyme, but it isseparated from hexose oxidase in this purification step.

[0076] 1.1.6. Mono Q Anion Exchange

[0077] The above pool resulting from the above phenyl sepharosechromatography step was desalted by ultrafiltration as described above.2 ml of this pool was applied to a Mono Q HR 5/5 column equilibrated in20 mM triethanolamine, pH 7.3. The column was subsequently eluted usinga 45 ml linear gradient from 0 to 0.65 M NaCl in equilibration buffer ata flow rate 1.5 ml/minute. Fractions of 1.5 ml were collected andfractions 14-24 were pooled.

[0078] 1.1.7. Mono P Anion Exchange

[0079] The hexose oxidase-containing pool from the above step 1.1.6 wasapplied to a Mono P HR 5/5 column equilibrated in 20 mM bis-Tris buffer,pH 6.5. The enzyme was eluted using a 45 ml linear gradient from 0 to0.65 M NaCl in equilibration buffer at a flow rate of 1.5 ml/minute, andfractions of 0.75 ml were collected. The highest hexose oxidase activitywas found in fraction 12.

[0080] b 1.2. Characterization of the Purified Hexose Oxidase

[0081] The hexose oxidase-containing pools from the above steps 1.1.6and 1.1.7 were used in the below characterization experiments:

[0082] 1.2.1. Determination of Molecular Weight

[0083] The size of the purified native hexose oxidase was determined bygel permeation chromatography using a Superose 6 HR 10/30 column at aflow rate of 0.5 ml/minute in 50 mM sodium phosphate buffer, pH 6.4.Ferritin (440 kD), catalase (232 kD), aldolase (158 kD), bovine serumalbumin (67 kD) and chymotrypsinogen (25 kD) were used as sizestandards. The molecular weight of the purified hexose oxidase wasdetermined to be 120±10 kD.

[0084] 1.2.2. Determination of pH Optimum

[0085] Assay mixtures for the determination of pH optimum (final volume300 μl) contained 120 μl of 0.1 M stock solution of sodiumphosphate/citrate buffer of varying pH values. All other assay mixturecomponents were dissolved in H₂O. The pH was determined in the dilutedstock buffer solutions at 25° C.

[0086] The hexose oxidase showed enzymatic activity from pH 3 to pH 8,but with optimum in the range of 3.5 to 5.5.

[0087] 1.2.3. K_(m) of the Hexose Oxidase for Glucose and Maltose,Respectively

[0088] Kinetic data were fitted to v=V_(max)S/(K_(m)+S), where V_(max)is the maximum velocity, S is the substrate concentration and K_(m) isthe concentration giving 50% of the maximum rate (Michaelis constant)using the EZ-FIT curve fitting microcomputer programme (Perrella, F. W.,1988, Analytical Biochemistry, 174:437-447).

[0089] A typical hyperbolic saturation curve was obtained for the enzymeactivity as a function of glucose and maltose, respectively. K_(m) forglucose was calculated to be 2.7 mM±0.7 mM and for maltose the K_(m) wasfound to be 43.7±5.6 mM.

EXAMPLE 2

[0090] Dough Improving Effect of Hexose Oxidase Extracted from Chondruscrispus

[0091] 2.1. Purification of Hexose Oxidase from Chondrus crispus

[0092] For this experiment, hexose oxidase was prepared in the followingmanner:

[0093] Fresh Chondrus crispus material was collected at the coast ofBrittany, France. The material was freeze-dried and subsequently ground.40 g of this ground material was suspended in 1000 ml of 20 mMtriethanolamine (TEA) buffer, pH 7.3 and left to stand at 5° C. forabout 64 hours with gentle agitation and then centrifuged at 2000×g for10 minutes. The supernatant was filtered through GF/A and GF/C glassfilters followed by filtering through a 45 μm pore size filter to obtaina filtrate preparation of 800 ml having hexose oxidase activitycorresponding to a glucose oxidase activity of 0.44 units per g ofpreparation. The activity was determined using the below procedure.

[0094] The supernatant was applied onto a 330 ml bed volumechromatographic column with anionic exchange Q Sepharose Big Beads (deadvolume 120 ml). The bound proteins were eluted over 180 minutes using agradient from 0 to 0.5 M NaCl in 20 mM TEA buffer, pH 7.3 followed by 1M NaCl in 20 mM TEA buffer, and fractions of 9 ml were collected andanalyzed for hexose oxidase activity using the below analyticalprocedure.

[0095] Hexose oxidase activity-containing fractions 60-83 were pooled(about 250 ml) and concentrated and desalted by ultrafiltration to about25 ml. This step was repeated twice on the retentates to which was added100 ml 0.05 mM TEA. The resulting retentate of 25 ml contained 0.95glucose oxidase activity units per g.

[0096] 2.2. Determination of Glucose Oxidase Activity

[0097] Definition: 1 glucose oxidase (GOD) unit corresponds to theamount of enzyme which under the specified conditions results in theconversion of 1 μmole glucose per min. The activity is stated as unitsper g of enzyme preparation.

[0098] Reagents: (i) Buffer: 20 g Na₂HPO₄-2H₂O is dissolved in 900 mldistilled water, pH is adjusted to 6.5; (ii) dye reagent (stocksolution): 200 mg of 2,6-dichloro-phenol-indophenol, Sigma No. D-1878-is dissolved in 1000 ml distilled water under vigorous agitation for 1hour; (iii) peroxidase (stock solution): Boehringer Mannheim No. 127361, 10,000 units is dissolved in 10 ml distilled water and 4.2 g ofammonium sulphate added; (iv) substrate: 10% w/v D-glucose solution inbuffer, (v) standard enzyme: hydrase #1423 from Amano.

[0099] Analytical principle and procedure: Glucose is converted togluconic acid and H₂O₂ which is subsequently converted by peroxidase toH₂O and O₂. The generated oxygen oxidizes the blue dye reagent2,6-dichloro-phenol-indophenol which thereby changes its colour topurple. The oxidized colour is measured spectrophotometrically at 590 nmand the enzymatic activity values calculated relative to a standard.

[0100] 2.3. The Effect of the Hexose Oxidase Preparation onCross-linking between Thiol Groups in a Wheat Flour Based Dough

[0101] The effect of hexose oxidase on the formation of thiol groupcross-linking was studied by measuring the content of free thiol groupsin a dough prepared from 1500 g of wheat flour, 400 Brabender Units (BU)of water, 90 g of yeast, 20 g of sucrose and 20 g of salt to which wasadded 0, 100, 250, 875 and 1250 units per kg of flour, respectively ofthe above hexose oxidase preparation. The measurement was carried outessentially in accordance with the colorimetric method of Ellman (1958)as also described in Cereal Chemistry, 1983, 70, 22-26. This method isbased on the principle that 5.5′-dithio-bis(2-nitrobenzoic acid) (DTNB)reacts with thiol groups in the dough to form a highly coloured anion of2-nitro-5-mercapto-benzoic acid, which is measuredspectrophotometrically at 412 nm.

[0102] Assuming that the relative change of the amount of thiol groupsin a dough is reflected as the change in the optical density (OD)resulting from-the reaction between thiol groups and DTNB in the dough,the following results were obtained: Hexose oxidase GOD units/kg flourOD₄₁₂ 0 0.297 100 0.285 250 0.265 875 0.187 1250 0.138

[0103] Thus, this experiment showed a significant decrease in ODindicating a reduction of the content of free thiol groups which wasproportionate to the amount of hexose oxidase activity added.

[0104] 2.4. Improvement of the Rheological Characteristics of Dough bythe Addition of Hexose Oxidase

[0105] The above dough was subjected to extensigraph measurementsaccording to AACC Method 54-10 with and without the addition of anamount of the hexose oxidase preparation corresponding to 100 units/kgflour of hexose oxidase activity. The dough without addition of enzymeserved as a control.

[0106] The principle of the above method is that the dough after formingis subjected to a load-extension test after resting at 30° C. for 45,90, 135 and 180 minutes, respectively, using an extensigraph capable ofrecording a load-extension curve (extensigram) which is an indication ofthe doughs resistance to physical deformation when stretched. From thiscurve, the resistance to extension, B (height of curve) and theextensibility, C (total length of curve) can be calculated. The B/Cratio (D) is an indication of the baking strength of the flour dough.

[0107] The results of the experiment is summarized in Table 2.1 below.TABLE 2.1 Extensigraph measurements of dough supplemented with 100 GODunits/kg flour of hexose oxidase (HOX). Sample Time, min B C D = B/CControl 45 230 180 1.3 HOX 45 320 180 1.8 Control 90 290 161 1.8 HOX 90450 148 3.0 Control 135 290 167 1.7 HOX 135 490 146 3.4 Control 180 300168 1.8 HOX 180 500 154 3.2

[0108] It is apparent from this table that the addition of hexoseoxidase (HOX) has an improving effect on the doughs resistance toextension as indicated by the increase in B-values. This is reflected inalmost a doubling of the B/C ratio as a clear indication that the bakingstrength of the flour is significantly enhanced by the hexose oxidaseaddition.

[0109] In a similar experiment, 100 units/kg flour of a commercialglucose oxidase product was added and the above parameters measured inthe same manner using a dough without enzyme addition as a control. Theresults of this experiment is shown in Table 2.2 below: TABLE 2.2Extensigraph measurements of dough supplemented with 100 GOD units/kgflour of glucose oxidase (GOX). Sample Time, min B C D = B/C Control 45240 180 1.3 GOX 45 290 170 1.7 Control 90 260 175 1.5 GOX 90 360 156 2.3Control 135 270 171 1.6 GOX 135 420 141 3.0

[0110] When the results for the above two experiments are compared withregard to differences between control dough and the hexose oxidase orglucose oxidase supplemented doughs it appeared that hexose oxidase hasa stronger strengthening effect than glucose oxidase. Furthermore, theB/C ratio increased more rapidly with hexose oxidase relative to glucoseoxidase which is a clear indication that enhancement of the bakingstrength is being conferred more efficiently by hexose oxidase than byglucose oxidase (FIG. 1 +L).

EXAMPLE 3

[0111] Dough Improving Effect of Hexose Oxidase Extracted from ChondrusCrispus

[0112] For this experiment fresh Chondrus crispus seaweed fronds wereharvested along the coast of Hirsholmene, Denmark. Hexose oxidase wasisolated using two different extraction procedures, and the materialsfrom both were pooled for the below dough improving experiment.

[0113] 3.1. Purification of Hexose Oxidase from Chondrus Crispus I

[0114] 954 g of the fresh fronds was rinsed in distilled water, driedwith a towel and stored in liquid nitrogen. The seaweed was blendedusing a Waring blender and 1908 ml of 0.1 M sodium phosphate buffer, 1 MNaCl, pH 6.8 was added to the blended seaweed. The mixture was extractedunder constant stirring for 4 days at 5° C., followed by centrifugationof the mixture at 20,000×g for 30 minutes.

[0115] The resulting 1910 ml supernatant (351.1 U/ml) was concentratedto 440 ml at 40° C. in a Büchi Rotavapor R110. The concentrate wasammonium sulphate fractionated to 25%. The mixture was stirred for 30minutes and centrifuged for 20 minutes at 47,000×g. The supernatant (395ml) was dialysed overnight against 20 l of 10 mM triethanolamine (TEA)buffer, pH 7.3 to a final volume of 610 ml (367.1 U/ml).

[0116] The above 610 ml was applied in two runs to a 2.6×25 cm columnwith 130 ml Q-Sepharose FF equilibrated in 20 mM TEA buffer, pH 7.3. Thecolumn was washed with the equilibration buffer and the bound proteinswere eluted using 800 ml gradient from 0 to 0.8 M NaCl in equilibrationbuffer. The column was eluted at 4 ml/minute and fractions of 12 mlcollected. Fractions containing the hexose oxidase activity werecollected and pooled to a final volume of 545 ml (241.4 U/ml).

[0117] 3.2. Purification of Hexose-Oxidase from Chondrus Crispus II

[0118] 1250 g of the fresh fronds was rinsed in distilled water, driedwith a towel and stored in liquid nitrogen. The seaweed was blended in aWaring blender followed by the addition of 2500 ml 0.1 M sodiumphosphate buffer, 1 M NaCl pH 6.8. The mixture was extracted undercontinuous stirring for 4 days at 5° C. followed by centrifugation at20,000×g for 30 minutes.

[0119] The resulting 2200 ml supernatant (332.8 U/ml) was concentratedto 445 ml at 40° C. using a Buchi Rotavapor R110. The resultingconcentrate was ammonium sulphate fractionated to 25%. The mixture wasstirred for 30 minutes and centrifuged for 20 minutes at 47,000×g. Theprecipitate was discarded. The 380 ml supernatant was dialysed overnightagainst 20 l 10 mM TEA buffer, pH 7.3, to a final volume of 850 ml(319.2 U/ml).

[0120] The above 850 ml was applied to a 2.6×25 cm column with 130 mlQ-Sepharose FF equilibrated in 20 mM TEA buffer, pH 7.3. The column waswashed with the equilibration buffer and the bound proteins were elutedusing 800 ml gradient from 0 to 0.8 M NaCl in equilibration buffer. Thecolumn was eluted at 4 ml/minute and fractions of 12 ml collected.Fractions containing the hexose oxidase activity were collected andpooled to a final volume of 288 ml.

[0121] The retentate from the above step was applied to a 2.6×31 cmcolumn with 185 ml metal chelating sepharose FF loaded with Ni²⁺ andequilibrated in 50 mM sodium phosphate, 1 M NaCl, pH 7.4. The boundproteins were eluted with a 740 ml gradient of 0 to 35 mM imidazole, pH4.7 in equilibration buffer. The column was eluted at 2 ml/minute andfractions of 11 ml was collected. Fractions 41-54 (140 ml, 352.3 U/ml)were pooled. Some hexose oxidase did run through the column.

[0122] 3.3. Pooling and Concentrating of Extracts

[0123] The run through and the 140 ml from purification II and the 545ml from purification I were pooled to a final volume of 1120 ml (303.6U/ml). The 1120 ml was rotation evaporated into a volume of 210 mlfollowed by dialysis overnight against 20 l of 10 mM TEA buffer, pH 7.3,to a final volume of 207 ml (1200.4 U/ml).

[0124] 3.3.1. Anion Exchange Step

[0125] The retentate resulting from the above step was applied to a2.6×25 cm column with 130 ml Q-sepharose FF equilibrated in 20 mMtriethanolamine, pH 7.3. The column was washed with the equilibrationbuffer and the bound proteins eluted using 800 ml gradient from 0 to 0.8M NaCl in equilibration buffer. The column was eluted at 4 ml/minute andfractions of 12 ml collected. Fractions 30-50 containing the hexoseoxidase activity (260 ml, 764.1 U/ml) were collected and pooled.

[0126] 3.3.2. Other Enzyme Activity

[0127] The above pooled solution was tested for the following enzymaticside activities catalase, protease, xylanase, α- and β-amylase andlipase. None of these activities were found in the solution.

[0128] 3.4. Improvement of the Rheological Characteristics of Dough bythe Addition of Hexose Oxidase

[0129] A dough was prepared from wheat flour, water and salt and 0, 72,216 and 360 units per kg of flour, respectively of the above hexoseoxidase preparation was added hereto. The dough without addition ofenzyme served as a control. In addition two doughs were prepared towhich was added 216 and 360 units per kg of flour respectively, ofGluzyme, a glucose oxidase available from Novo Nordisk A/S, Denmark.

[0130] The doughs were subjected to extensigraph measurements accordingto a modification of the above AACC Method 54-10. The results of theexperiment are summarized in Table 3.1 below. TABLE 3.1 Extensigraphmeasurements of dough supplemented with hexose oxidase (HOX) or glucoseoxidase (units per kg flour) Time, Sample min. B C D = B/C Control 45250 158 1.6 HOX 72 U/kg 45 330 156 2.1 HOX 216 U/kg 45 460 153 3.0 HOX360 U/kg 45 580 130 4.5 Gluzyme 72 U/kg 45 350 159 2.2 Gluzyme 216 U/kg45 340 148 2.3 Gluzyme 360 U/kg 45 480 157 3.1 Control 90 290 164 1.8HOX 72 U/kg 90 470 145 3.2 HOX 216 U/kg 90 650 142 4.6 HOX 360 U/kg 90870 116 7.5 Gluzyme 72 U/kg 90 450 147 3.1 Gluzyme 216 U/kg 90 480 1383.5 Gluzyme 360 U/kg 90 500 152 3.2 Control 135 330 156 2.1 HOX 72 U/kg135 540 129 4.2 HOX 216 U/kg 135 750 125 6.0 HOX 360 U/kg 135 880 1177.5 Gluzyme 72 U/kg 135 510 136 3.8 Gluzyme 216 U/kg 135 550 122 4.5Gluzyme 360 U/kg 135 560 121 4.6

[0131] It is evident from the above table that the addition of hexoseoxidase (HOX) or glucose oxidase had an improving effect on theresistance of doughs to extension as indicated by the increase inB-values. This is reflected in an increase of the B/C ratio as a clearindication that the baking strength of the flour was enhancedsignificantly by the addition of enzymes.

[0132] It is also evident that the hexose oxidase had a higherstrengthening effect than glucose oxidase. Furthermore, the B/C ratioincreased more rapidly with hexose oxidase relative to glucose oxidasewhich is a clear indication that enhancement of the baking strength isbeing conferred more efficiently by hexose oxidase than by glucoseoxidase.

EXAMPLE 4

[0133] Dough Improving Effect of Hexose Oxidase Extracted from ChondrusCrispus

[0134] 4.1. Purification of Hexose Oxidase from Chondrus Crispus

[0135] Fresh Chondrus crispus fronds were harvested along the coast ofBrittany, France. 2285 g of this fresh material was rinsed in distilledwater, dried with a towel and stored in liquid nitrogen. The seaweed wasblended in a Waring blender followed by addition of 4570 ml 0.1 M sodiumphosphate buffer, 1 M NaCl pH 6.8. The mixture was extracted undercontinuous magnetic stirring for 4 days at 5° C. followed bycentrifugation at 20,000×g for 30 minutes.

[0136] The resulting 4930 ml supernatant (624.4 U/ml) was concentratedto 1508 ml at 40° C. using a Büchi Rotavapor R110. The obtainedconcentrate was polyethylenglycol fractionated to 3% (w/v). The mixturewas stirred for 30 minutes and centrifuged for 30 minutes at 47,000×g.The pellet was discarded. The 1470 ml supernatant (2118.7 U/ml) was PEGfractionated to 24%. The mixture was stirred for 30 minutes andcentrifuged for 30 minutes at 47,000×g. The supernatant was discardedand the 414.15 g of precipitate was resuspended in 200 ml 20 mM TEAbuffer, pH 7.3, followed by dialysis over night at 5° C. against 20 l 10mM TEA buffer, pH 7.3.

[0137] After dialysis the volume was 650 ml (2968.6 U/ml). Thesuspension was centrifuged for 30 minutes at 20,000×g. The precipitatewas discarded and the supernatant was diluted to 3200 ml with distilledwater.

[0138] The above 3200 ml (829.9 U/ml) was applied to a 10×14 cm columnwith 1100 ml Q-Sepharose FF equilibrated in 20 mM TEA buffer, pH 7.3.The column was washed with the equilibration buffer and the boundproteins were eluted using 15,000 ml gradient from 0 to 0.8 M NaCl inequilibration buffer. The column was eluted at 50 ml/minute. Hexoseoxidase did run through the column and 840 ml of this was collected.

[0139] The 840 ml suspension was treated with kieselguhr andconcentrated to 335 ml (2693.3 U/ml).

[0140] The above 335 ml was applied to a 3 l Sephadex G25C desaltingcolumn 10×40 cm. The column was equilibrated in 20 mM TEA buffer, pH7.3, eluted at a flow rate of 100 ml/minute and 970 ml eluate wascollected. This eluate was applied to a 10×14 cm column with 1100 mlQ-Sepharose FF equilibrated in 20 mM TEA, pH 7.3. The column was washedwith the equilibration buffer and bound proteins eluted using a 15,000ml gradient of 0 to 0.8 M NaCl in equilibration buffer. The column waseluted at 50 ml/min. Hexose oxidase did run through the column and 1035ml of this was collected.

[0141] To the above eluate (1035 ml) ammonium sulphate was added to afinal concentration of 2 M. The mixture was then applied in two runs toa 5×10 cm column with 200 ml phenyl sepharose HP equilibrated in 25 mMsodium phosphate buffer, pH 6.3 and 2 M (NH₄)₂SO₄. The column was washedwith equilibration buffer followed by eluting the bound proteins at aflow rate of 50 ml/minute using 5,000 ml gradient from 2 M to 0 M(NH₄)₂SO₄ in 25 mM sodium phosphate buffer. Fractions of 500 and 29 ml,respectively were collected from run 1 and 2. Fraction 5 in run 1 andfractions 27-42 in run 2 containing the hexose activity were pooled to atotal of 1050 ml (563.9 U/ml).

[0142] The above pool was desalted by a 3 l Sephadex G25C gel filtrationcolumn. The column was equilibrated in 20 mM TEA buffer, pH 7.3, elutedat a flow rate of 100 ml/minute and 1,000 ml eluate was collected.

[0143] The 1,000 ml eluate was concentrated to 202 ml (2310.2 U/ml) andthis preparation was used for following rheology testing.

[0144] 4.2. Improvement of the Rheological Characteristics of Dough bythe Addition of Hexose Oxidase

[0145] A dough was prepared from wheat flour, water and salt and 0, 288,504 and 720 oxidoreductase units per kg of flour, respectively of theabove hexose oxidase preparation was added hereto. The dough withoutaddition of enzyme served as a control. In addition two doughs wereprepared to which was added 288 and 504 oxidoreductase units per kg offlour respectively, of Gluzyme, a glucose oxidase available from NovoNordisk A/S, Denmark.

[0146] The doughs were subjected to extensigraph measurements accordingto a modification of AACC Method 54-10.

[0147] The results of the experiment are summarized in Table 4.1 below.TABLE 4.1 Extensigraph measurements of dough supplemented with hexoseoxidase (HOX) or glucose oxidase (Units per kg flour) Time, Sample min.B C D = B/C Control 45 210 171 1.2 HOX 288 U/kg 45 490 139 3.5 HOX 504U/kg 45 640 122 5.2 HOX 720 u/kg 45 730 109 6.7 Gluzyme 288 U/kg 45 350165 2.1 Gluzyme 504 U/kg 45 385 153 2.5 Gluzyme 720 U/kg 45 435 148 2.9Control 90 275 182 1.5 HOX 288 U/kg 90 710 130 5.5 HOX 504 U/kg 90 825106 7.8 HOX 720 U/kg 90 905 107 8.5 Gluzyme 288 U/kg 90 465 153 3.0Gluzyme 504 U/kg 90 515 135 3.8 Gluzyme 720 U/kg 90 540 140 3.9 Control135 280 175 1.6 HOX 288 U/kg 135 745 102 7.3 HOX 504 U/kg 135 920 94 9.8HOX 720 U/kg 135 — 80 — Gluzyme 288 U/kg 135 525 129 4.1 Gluzyme 504U/kg 135 595 129 4.6 Gluzyme 720 U/kg 135 630 121 5.2

[0148] It is apparent from the above results that the addition of hexoseoxidase (HOX) or glucose oxidase has an improving effect on theresistance of doughs to extension as indicated by the increase inB-values. This is reflected in an increase of the B/C ratio.

[0149] It is also apparent that hexose oxidase has a strongerstrengthening effect than that of glucose oxidase, the strengtheningeffect of both enzymes being proportional to the amount of enzyme added.Furthermore, the B/C ratio increased more rapidly with hexose oxidaserelative to glucose oxidase which is a clear indication that enhancementof the baking strength is being conferred more efficiently by hexoseoxidase than by glucose oxidase.

EXAMPLE 5

[0150] Improving Effect of Hexose Oxidase Extracted from Chondruscrispus on the Specific Volume of Bread

[0151] 5.1. Purification of Hexose Oxidase from Chondrus crispus

[0152] Fresh Chondrus crispus fronds were harvested along the coast ofBrittany, France. 2191 g of this fresh material was rinsed in distilledwater, dried with a towel and stored in liquid nitrogen. The seaweed wasblended in a Waring blender followed by addition of 4382 ml 0.1 M sodiumphosphate buffer, 1 M NaCl and pH 6.8. The mixture was extracted undercontinuously magnetic stirring for 4 days at 5° C. followed bycentrifugation at 20,000×g for 20 minutes.

[0153] The resulting 4600 ml supernatant (746.1 U/ml) was concentratedto 850 ml at 40° C. in a Büchi Rotavapor R110. This concentrate (3626.9U/ml) was polyethylene glycol fractionated to 3% (w/v). The mixture wasstirred for 30 minutes and centrifuged for 30 minutes at 20,000×g. Theprecipitate was discarded. The 705 ml supernatant (2489.8 U/ml) was PEGfractionated to 25%. The mixture was stirred for 30 minutes andcentrifuged for 30 minutes at 20,000×g. The supernatant was discardedand the 341 g of precipitate was resuspended in 225 ml 20 mM TEA buffer,pH 7.3. The suspension (500 ml) was desalted on a 3 l Sephadex G25Cdesalting column 10×40 cm. The column was equilibrated in 20 mM TEAbuffer, pH 7.3, and eluted at a flow rate of 100 ml/minute. 1605 mleluate was collected.

[0154] To the above eluate (687.5 U/ml) ammonium sulphate was added to afinal concentration of 2 M. The mixture was then applied in two runs toa 5×10 cm column with 200 ml phenyl sepharose HP equilibrated in 25 mMsodium phosphate buffer, pH 6.3 and 2 M (NH₄)₂SO₄. The column was washedwith equilibration buffer followed by elution of the bound proteins at aflow rate of 50 ml/minute using 5,000 ml gradient from 2 M to 0 M(NH₄)₂SO₄ in 25 mM sodium phosphate buffer. Fractions of 29 ml wascollected. Fractions 85-105 in run 1 and fractions 36-69 in run 2containing the hexose activity were pooled to a total of 1485 ml (194.7U/ml).

[0155] The above pool was desalted by a 3 l Sephadex G25C gel filtrationcolumn, the same as used in 4.1. The column was equilibrated in 20 mMTEA buffer, pH 7.3, and eluted at a flow rate of 100 ml/minute. 1,200 mleluate was collected.

[0156] The 1,200 ml eluate was concentrated to 685 ml (726.2 U/ml) andused for baking experiments.

[0157] 5.2. Improvement of the Specific Volume of Bread by Adding HexoseOxidase to the Dough

[0158] A dough was prepared from 1500 g of flour, 90 g of yeast, 24 g ofsalt, 24 g of sugar and 400 BU of water and 0 or 108 units of the abovepurified hexose oxidase and 108 units of Gluzyme (glucose oxidaseavailable from Novo Nordisk, Denmark) per kg flour, respectively wasadded hereto. The dough was mixed on a Hobart mixer for 20+9 minutes at26° C. and divided into two parts followed by resting for 10 minutes at30° C. in a heating cabinet, moulding with a Fortuna 3/17/7 and proofingfor 45 minutes at 34° C. and 85% RH. The thus proofed dough was baked at220° C. for 17 minutes with 12 sec. steam in a Bago oven.

[0159] The results of the experiment are summarized in table 5.1 below.TABLE 5.1 Improvement of specific volumes of bread prepared from doughsupplemented with hexose oxidase or glucose oxidase (Units per kg flour)Total Total Specific volume weight volume Control 5325 1027 5.18 Hexoseoxidase 108 U/kg 6650 1036 6.41 Gluzyme 108 U/kg 6075 1030 5.89

[0160] It is evident from the above table that the addition of hexoseoxidase or glucose oxidase had an increasing effect on the total volume,the weight being essentially the same. This is reflected in an increaseof the specific volume as compared to the bread baked without additionof enzymes.

[0161] It is also evident that hexose oxidase has a significantly largereffect on the increase of the specific volume than had glucose oxidaseat the same dosage.

EXAMPLE 6

[0162] Characterization of the Purified Hexose Oxidase

[0163] Preparations from the above purifications were used forcharacterization of hexose oxidase.

[0164] 6.1. Staining for Hexose Activity After Non-denaturing PAGE

[0165] Hexose oxidase activity was analyzed by native PAGE using precast8-16 % Tris-glycine Novex gels according to the manufacturesinstructions (Novex, San Diego, USA). After electrophoresis the gelswere stained for hexose activity by incubation of the gel in a solutioncontaining 50 mM sodium phosphate buffer, pH 6.0, 100 mM glucose, 50mg/l phenazine methosulphate (Sigma P9625) and 250 mg/l nitrobluetetrazolium (Sigma N6876) as described in the PhD thesis by Witteveen,C. F. B. (1993) “Gluconate formation and polyol metabolism inAspergillus niger”. After about 30 minutes the hexose activity wasvisible as a double bond very close to each other. The same double bandwas also seen when a native PAGE of hexose oxidase was silver stained.The molecular weight of purified hexose oxidase was determined to 144 kDby native PAGE. Half the gel was silver stained, the other half wasactivity stained. As standards were used bovine serum albumin (67 kD),lactate dehydrogenase (140 kD), catalase (232 kD), ferritin (440 kD) andthyroglobulin (669 kD).

[0166] 6.2 Determination of Molecular Weight by SDS-Page

[0167] The molecular weight was also determined on material which wasfirst applied to a native PAGE as described above, after activitystaining the hexose oxidase band was excised from the gel and thenelectroeluted using an Electro-Eluter (model 422, Bio-Rad, CA, USA)according to the manufacturer's recommendations. The electroelutedprotein was subjected to SDS-PAGE and silver stained. This material gave“one” double bond at about 70 kDa in SDS-PAGE gels. The electroelutedhexose oxidase is therefore a dimer of two subunits.

[0168] 6.3 Determination of pI of Hexose Oxidase

[0169] Samples containing hexose oxidase activity were analyzed byisoelectric focusing (IEF) using a precast 3-10 IEF gel according to themanufacturer's recommendations (Novex, San Diego, US). Afterelectrophoresis half of the gel was silver stained and the other halfnitroblue tetrazolium stained as described in 6.1.

[0170] Hexose oxidase stained as a double band. The pI of the first bandwas 4.79, pI of the second band was 4.64. As standards were usedtrypsinogen (9.30), lentil lectin basic band (8.65), lentil lectinmiddle band (8.45), lentil lectin acid band (8.15), horse myoglobinacidic band (6.85), human carbonic anhydrase B (5.85), β-lactoglobulin A(5.20), soy bean trypsin inhibitor (4.55) and amyloglucosidase (3.50).

[0171] 6.4 Determination of K_(m) of Hexose Oxidase for Different Sugars

[0172] K_(m) of hexose oxidase was determined for 7 different sugars asdescribed in 1.2.3. Results are summarized in table 6.1 below. TABLE 6.1Determination of K_(m) of hexose oxidase for different sugars SubstrateK_(m) (mM) CV (mM) D-glucose 2.7 0.7 D-galactose 3.6 1 cellobiose 20.27.8 maltose 43.7 5.6 lactose 90.3 20.6 xylose 102 26 arabinose 531 158

[0173] 6.5 Determination of a Peptide Sequence of the Hexose Oxidase

[0174] 50 μl from the electroeluted mixture in 6.2 was suspended in 450μl 0.1% triflouracetic acid (TFA).

[0175] To remove the Tris, glycine and SDS, the above mixture wassubjected to chromatography on reverse-phase HPLC. The resultingsolution was applied in 9 runs to a 4.6×30 cm Brownlee C2 columnequilibrated in 0.1 % TFA. The column was washed in equilibration bufferand bound peptides eluted with a 14 ml gradient from 10 to 80 %acetonitrile in 0.1% TFA, at a flow rate of 0.7 ml/min. Fractions fromthe largest peak containing the enzyme were collected and freeze dried.

[0176] 6.5.1 Endoproteinase Lys-C Digestion

[0177] The resulting freeze dried enzyme was dissolved in 50 μl 8 Murea, 0.4 M NH₄HCO₃, pH 8.4. Denaturation and reduction of the proteinwas carried out by the addition of 5 μl mM dithiothreitol and under anoverlay of N₂ at 50° C. for 15 min. The solution was cooled to roomtemperature and 5 μl 100 mM iodoacetamide was added, the cysteines beingderivatized for 15 min. at room temperature in the dark under N₂.Subsequently, the solution was suspended in 135 μl water and digestionwas carried out at 37° C. under N₂ for 24 hours by addition of 5 μgendoproteinase Lys-C dissolved in 5 μl water. The reaction wasterminated by freezing the reaction mixture at −20° C.

[0178] 6.5.2 Reverse-phase HPLC Separation of Peptides

[0179] The resulting peptides were separated by reverse-phase HPLC on aVYDAC C18 column 0.46×15 cm (The Separation Group, CA, USA) using assolvent A 0.1 % TFA in water and as solvent B 0.1 % TFA in acetonitrile.

[0180] 6.5.3 Peptide Sequencing

[0181] Sequencing was performed on an Applied Biosystems 476A sequencer(Applied Biosystems, CA, USA) using pulsed-liquid fast cycles accordingto the manufacturer's instructions. A peptide having the below aminoacid sequence was identified:

[0182] D P G Y I V I D V N A G T P D K P D P.

1. A method of improving the rheological properties of a flour dough and the quality of the finished product made from the dough, comprising adding to the dough ingredients, dough additives or the dough an effective amount of an oxidoreductase which is at least capable of oxidizing maltose.
 2. A method according to claim 1 wherein the oxidoreductase is hexose oxidase.
 3. A method according to claim 2 wherein the hexose oxidase is derived from a source selected from an algal species, a plant species and a microbial species.
 4. A method according to claim 3 wherein the hexose oxidase is derived from Chondrus crispus.
 5. A method according to claim 2 wherein hexose oxidase is 15 added in an amount which is in the range of 1 to 10,000 units per kg of flour.
 6. A method according to claim 5 wherein the hexose oxidase is added in an amount which is in the range of 10 to 1000 units per kg of flour.
 7. A method according to claim 1 or 2 wherein the resistance to extension of the dough in terms of the ratio between the resistance to extension (height of curve, B) and the extensibility (length of curve, C), i.e. the B/C ratio, as measured by the AACC method 54-10 is increased by at least 10% relative to that of an otherwise similar dough not containing oxidoreductase.
 8. A method according to claim 1 wherein the finished product is bread.
 9. A method according to claim 1 wherein the finished product is a noodle product.
 10. A method according to claim 1 wherein the finished product is an alimentary paste product.
 11. A method according to claim 1 wherein at least one further enzyme is added to the dough ingredients, dough additives or the dough.
 12. A method according to claim 11 wherein the further enzyme is selected from the group consisting of a cellulase, a hemicellulase, a xylanase, a starch degrading enzyme, a glucose oxidase, a lipase and a protease.
 13. A dough improving composition comprising an oxidoreductase which is at least capable of oxidizing maltose and at least one further dough ingredient or dough additive.
 14. A composition according to claim 13 wherein the oxidoreductase is derived from a source selected from an algal species, a plant species and a microbial species.
 15. A composition according to claim 14 wherein the oxidoreductase is hexose oxidase.
 16. A composition according to claim 15 wherein the hexose oxidase is derived from Chondrus crispus.
 17. A composition according to claim 13 which is a pre-mixture useful for preparing a baked product or in making a noodle product or an alimentary paste product.
 18. A composition according to claim 13 which comprises an additive selected from the group consisting of an emulsifying agent and a hydrocolloid.
 19. A composition according to claim 18 wherein the hydrocolloid is selected from the group consisting of an alginate, a carrageenan, a pectin and a vegetable gum.
 20. A method of preparing a bakery product the method comprising preparing a flour dough to which is added an effective amount of an oxidoreductase which is at least capable of oxidizing maltose, and baking the dough.
 21. A method according to claim 20 wherein the specific volume of the bakery product is increased relative to an otherwise similar bakery product prepared from a dough not containing oxidoreductase.
 22. A method according to claim 21 wherein the specific volume is increased by at least 20%.
 23. A method according to claim 20 wherein at least one further enzyme is added to the dough.
 24. A method according to claim 20 wherein the further enzyme is selected from the group consisting of a cellulase, hemicellulase, a xylanase, an starch degrading enzyme, a glucose oxidase, a lipase and a protease.
 25. A method according to claim 20 wherein the oxidoreductase is hexose oxidase.
 26. A method of preparing a flour dough-based food product, comprising adding to the dough an effective amount of a maltose oxidizing oxidoreductase.
 27. A method according to claim 26 wherein the oxidoreductase is hexose oxidase. 